1. Introduction
Appropriate
antimicrobial drug use has unquestionable benefit, but physicians and the
public frequently use these agents inappropriately. Inappropriate use results
from physicians providing antimicrobial drugs to treat viral infections, using
inadequate criteria for diagnosis of infections that potentially have a
bacterial aetiology, unnecessarily prescribing expensive, broad-spectrum
agents, and not following established recommendations for using chemo
prophylaxis. The availability of antibiotics over the counter, despite
regulations to the contrary, also fuel inappropriate usage of antimicrobial
drugs in India. The easy availability of antimicrobial drugs leads to their
incorporation into herbal or "folk" remedies, which also increases
inappropriate use of these agents.
Widespread antibiotic
usage exerts a selective pressure that acts as a driving force in the
development of antibiotic resistance. The association between increased rates
of antimicrobial use and resistance has been documented for nosocomial
infections as well as for resistant community acquired infections. As
resistance develops to "first-line" antibiotics, therapy with new,
broader spectrum, more expensive antibiotics increases, but is followed by
development of resistance to the new class of drugs.
Resistance factors,
particularly those carried on mobile elements, can spread rapidly within human
and animal populations. Multidrug-resistant pathogens travel not only locally
but also globally, with newly introduced pathogens spreading rapidly in
susceptible hosts. Antibiotic resistance patterns may vary locally and
regionally, so surveillance data needs to be collected from selected sentinel sources.
Patterns can change rapidly and they need to be monitored closely because of
their implications for public health and as an indicator of appropriate or
inappropriate antibiotic usage by physicians in that area.
The results of
in-vitro antibiotic susceptibility testing, guide clinicians in the appropriate
selection of initial empiric regimens and, drugs used for individual patients
in specific situations. The selection of an antibiotic panel for susceptibility
testing is based on the commonly observed susceptibility patterns, and is
revised periodically.
2. Principle
The principles
of determining the effectivity of a noxious agent to a bacterium were well
enumerated by Rideal ,Walker and others at the turn of the century, the
discovery of antibiotics made these tests(or their modification)too cumbersome
for the large numbers of tests necessary to be put up as a routine. The ditch
plate method of agar diffusion used by Alexander Fleming was the forerunner of
a variety of agar diffusion methods devised by workers in this field .The
Oxford group used these methods initially to assay the antibiotic contained in
blood by allowing the antibiotics to diffuse out of reservoirs in the medium in
containers placed on the surface.
With the
introduction of a variety of antimicrobials it became necessary to perform the
antimicrobial susceptibility test as a routine.
For this, the antimicrobial contained in a reservoir was allowed to
diffuse out into the medium and interact in a plate freshly seeded with the
test organisms. Even now a variety of
antimicrobial containing reservoirs are used but the antimicrobial impregnated
absorbent paper disc is by far the commonest type used. The disc diffusion method of AST is the most
practical method and is still the method of choice for the average
laboratory. Automation may force the
method out of the diagnostic laboratory but in this country as well as in the
smaller laboratories of even advanced countries, it will certainly be the most
commonly carried out microbiological test for many years to come. It is,
therefore, imperative that microbiologists understand the principles of the
test well and keep updating the information as and when necessary. All
techniques involve either diffusion of antimicrobial agent in agar or dilution
of antibiotic in agar or broth.
Even automated
techniques are variations of the above methods.
3.
Factors Influencing Antimicrobial Susceptibility Testing
pH
The pH of each batch
of Müeller-Hinton agar should be
checked when the medium is prepared. The
exact method used will depend largely on the type of equipment available in the
laboratory. The agar medium should have a pH between 7.2 and 7.4 at room
temperature after gelling. If the pH is too low, certain drugs will appear to
lose potency (e.g., aminoglycosides, quinolones, and macrolides), while other
agents may appear to have excessive activity (e.g., tetracyclines). If the pH is too high, the opposite effects
can be expected. The pH can be checked
by one of the following means:
* Macerate
a sufficient amount of agar to submerge the tip of a pH electrode.
* Allow
a small amount of agar to solidify around the tip of a pH electrode in a beaker
or cup.
* Use
a properly calibrated surface electrode.
Moisture
If, just before use,
excess surface moisture is present, the plates should be placed in an incubator
(35°C)
or a laminar flow hood at room temperature with lids ajar until excess surface
moisture is lost by evaporation (usually 10 to 30 minutes). The surface should be moist, but no droplets
of moisture should be apparent on the surface of the medium or on the petri
dish covers when the plates are inoculated.
Effects of Thymidine or Thymine
Media containing
excessive amounts of thymidine or thymine can reverse the inhibitory effect of
sulfonamides and trimethoprim, thus yielding smaller and less distinct zones,
or even no zone at all, which may result in false-resistance reports. Müeller-Hinton
agar that is as low in thymidine content as possible should be used. To evaluate a new lot of Müeller-Hinton agar, Enterococcus faecalis ATCC 29212, or
alternatively, E. faecalis ATCC
33186, should be tested with trimethoprim/sulfamethoxazole disks. Satisfactory media will provide essentially
clear, distinct zones of inhibition 20 mm or greater in diameter. Unsatisfactory media will produce no zone of
inhibition, growth within the zone, or a zone of less than 20 mm.
Effects of Variation in Divalent Cations
Variation in divalent
cations, principally magnesium and calcium, will affect results of aminoglycoside
and tetracycline tests with P. aeruginosa
strains. Excessive cation content will
reduce zone sizes, whereas low cation content may result in unacceptably large
zones of inhibition. Excess zinc ions may reduce zone sizes of carbapenems. Performance tests with each lot of Müeller-Hinton agar must conform to the control
limits.
Testing strains that fail to grow satisfactorily
Only aerobic or
facultative bacteria that grow well on unsupplemented Müeller-Hinton agar
should be tested on that medium. Certain
fastidious bacteria such as Haemophilus
spp.,
N.
gonorrhoeae, S. pneumoniae, and viridans and
ß-haemolytic streptococci do not grow sufficiently on unsupplemented
Müeller-Hinton agar. These organisms
require supplements or different media to grow, and they should be tested on
the media described in separate sections.
4. Methods of Antimicrobial
Susceptibility Testing
Antimicrobial susceptibility testing
methods are divided into types based on the principle applied in each system.
They include:
Diffusion Dilution Diffusion&Dilution
Stokes method Minimum Inhibitory Concentration E-Test method
Kirby-Bauer method i)
Broth dilution
ii)Agar Dilution
4.1 Disk Diffusion
4.1 Disk Diffusion
Reagents
for the Disk Diffusion Test
1.
Müeller-Hinton Agar Medium
Of the many media available, Müeller-Hinton agar is
considered to be the best for routine susceptibility testing of nonfastidious
bacteria for the following reasons:
* It shows acceptable batch-to-batch
reproducibility for susceptibility testing.
* It is low in
sulphonamide, trimethoprim, and tetracycline inhibitors.
* It gives satisfactory growth of most
nonfastidious pathogens.
* A large body of data and experience
has been collected concerning susceptibility tests performed with this medium.
Although Müeller-Hinton agar is
reliable generally for susceptibility testing, results obtained with some
batches may, on occasion, vary significantly.
If a batch of medium does not support adequate growth of a test
organism, zones obtained in a disk diffusion test will usually be larger than
expected and may exceed the acceptable quality control limits. Only Müeller-Hinton medium formulations that
have been tested according to, and that meet the acceptance limits described
in, NCCLS document M62-A7- Protocols for Evaluating Dehydrated Müeller-Hinton
Agar should be used.
Preparation of Müeller-Hinton
Agar
Müeller-Hinton agar preparation includes the following steps.
1. Müeller-Hinton agar should be prepared
from a commercially available dehydrated base according to the manufacturer's
instructions.
2. Immediately after autoclaving, allow it
to cool in a 45 to 50°C water bath.
3. Pour the freshly prepared and cooled
medium into glass or plastic, flat-bottomed petri dishes on a level, horizontal
surface to give a uniform depth of approximately 4 mm. This corresponds to 60 to 70 ml of medium for
plates with diameters of 150 mm and 25 to 30 ml for plates with a diameter of
100 mm.
4. The agar medium should be allowed to
cool to room temperature and, unless the plate is used the same day, stored in
a refrigerator (2 to 8°C).
5. Plates should be used within seven days
after preparation unless adequate precautions, such as wrapping in plastic,
have been taken to minimize drying of the agar.
6. A representative sample of each batch
of plates should be examined for sterility by incubating at 30 to 35°C for 24 hours or longer.
2. Preparation of antibiotic stock solutions
Antibitiotics may be received as powders or tablets. It is recommended to
obtain pure antibiotics from commercial sources, and not use injectable
solutions. Powders must be accurately
weighed and dissolved in the appropriate diluents (Annexure III) to yield the
required concentration, using sterile glassware. Standard strains of stock cultures
should be used to evaluate the antibiotic stock solution. If satisfactory, the
stock can be aliquoted in 5 ml volumes and frozen at -20ºC or -60ºC.
Stock solutions are prepared using the formula (1000/P) X V X C=W, where
P+potency of the anitbiotic base, V=volume in ml required, C=final
concentration of solution and W=weight of the antimicrobial to be dissolved in
V.
Preparation of dried filter paper
discs
Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in
diameter, which are placed in a Petri dish and sterilized in a hot air oven.
The loop used for delivering the antibiotics is made of 20 gauge wire and
has a diameter of 2 mm. This delivers 0.005 ml of antibiotics to each disc.
Storage of commercial
antimicrobial discs
Cartridges containing commercially prepared paper disks specifically for
susceptibility testing are generally packaged to ensure appropriate anhydrous
conditions. Discs should be stored as
follows:
* Refrigerate the containers at 8°C or below, or freeze at -14°C or below, in a nonfrost-free freezer until needed. Sealed packages of disks that contain drugs
from the ß-lactam class should be stored frozen, except for a small working
supply, which may be refrigerated for at most one week. Some labile agents (e.g., imipenem, cefaclor,
and clavulanic acid combinations) may retain greater stability if stored frozen
until the day of use.
* The unopened disc containers should be
removed from the refrigerator or freezer one to two hours before use, so they
may equilibrate to room temperature before opening. This procedure minimizes the amount of
condensation that occurs when warm air contacts cold disks.
* Once a cartridge of discs has been
removed from its sealed package, it should be placed in a tightly sealed,
desiccated container. When using a
disc-dispensing apparatus, it should be fitted with a tight cover and supplied
with an adequate desiccant. The
dispenser should be allowed to warm to room temperature before opening. Excessive moisture should be avoided by replacing
the desiccant when the indicator changes color.
* When not in use, the dispensing
apparatus containing the discs should always be refrigerated.
* Only those discs that have not reached
the manufacturer's expiration date stated on the label may be used. Discs should be discarded on the expiration
date.
Turbidity standard for inoculum
preparation
To standardize the inoculum density for a susceptibility test, a BaSO4
turbidity standard, equivalent to a 0.5 McFarland standard or its optical
equivalent (e.g., latex particle suspension), should be used. A BaSO4 0.5 McFarland standard may
be prepared as follows:
1.
A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2
. 2H2O) is added to 99.5 ml of 0.18 mol/L H2SO4
(1% v/v) with constant stirring to maintain a suspension.
2.
The correct density of the turbidity standard should be verified by using
a spectrophotometer with a 1-cm light path and matched cuvette to determine the
absorbance. The absorbance at 625 nm
should be 0.008 to 0.10 for the 0.5 McFarland standard.
3.
The Barium Sulfate suspension should be transferred in 4 to 6 ml aliquots
into screw-cap tubes of the same size as those used in growing or diluting the
bacterial inoculum.
4.
These tubes should be tightly sealed and stored in the dark at room temperature.
5.
The barium sulfate turbidity standard should be vigorously agitated on a
mechanical vortex mixer before each use and inspected for a uniformly turbid
appearance. If large particles appear,
the standard should be replaced. Latex
particle suspensions should be mixed by inverting gently, not on a vortex mixer
6.
The barium sulfate standards should be replaced or their densities
verified monthly.
Disc diffusion methods
The Kirby-Bauer and Stokes'
methods are usually used for antimicrobial susceptibility testing, with the
Kirby-Bauer method being recommended by the NCCLS. The accuracy and
reproducibility of this test are dependent on maintaining a standard set of
procedures as described here.
NCCLS is an international, interdisciplinary, non-profit,
non-governmental organization composed of medical professionals, government,
industry, healthcare providers, educators etc. It promotes accurate
antimicrobial susceptibility testing (AST) and appropriate reporting by
developing standard reference methods, interpretative criteria for the results
of standard AST methods, establishing quality control parameters for standard
test methods, provides testing and reporting strategies that are clinically
relevant and cost-effective
Interpretative criteria of NCCLS
are developed based on international collaborative studies and well correlated
with MIC’s and the results have corroborated with clinical data. Based on study
results NCCLS interpretative criteria are revised frequently. NCCLS is approved
by FDA-USA and recommended by WHO.
Procedure for Performing the Disc
Diffusion Test
Inoculum
Preparation
Growth Method
The growth
method is performed as follows
1. At least three to five well-isolated
colonies of the same morphological type are selected from an agar plate
culture. The top of each colony is
touched with a loop, and the growth is transferred into a tube containing 4 to
5 ml of a suitable broth medium, such as tryptic soy broth.
2. The broth culture is incubated at 35°C until it achieves or exceeds the turbidity of the 0.5 McFarland
standard (usually 2 to 6 hours)
3. The turbidity of the actively growing
broth culture is adjusted with sterile saline or broth to obtain a turbidity
optically comparable to that of the 0.5 McFarland standard. This results in a suspension containing
approximately 1 to 2 x 108 CFU/ml for E.coli ATCC 25922. To
perform this step properly, either a photometric device can be used or, if done
visually, adequate light is needed to visually compare the inoculum tube and
the 0.5 McFarland standard against a card with a white background and
contrasting black lines.
Direct Colony Suspension Method
1. As a convenient alternative to the
growth method, the inoculum can be prepared by making a direct broth or saline
suspension of isolated colonies selected from a 18- to 24-hour agar plate (a
nonselective medium, such as blood agar, should be used). The suspension is adjusted to match the 0.5
McFarland turbidity standard, using saline and a vortex mixer.
2. This approach is the recommended method
for testing the fastidious organisms, Haemophilus
spp., N. gonorrhoeae, and
streptococci, and for testing staphylococci for potential methicillin or
oxacillin resistance.
Inoculation
of Test Plates
1. Optimally, within 15 minutes after
adjusting the turbidity of the inoculum suspension, a sterile cotton swab is
dipped into the adjusted suspension. The
swab should be rotated several times and pressed firmly on the inside wall of
the tube above the fluid level. This
will remove excess inoculum from the swab.
2. The dried surface of a Müeller-Hinton
agar plate is inoculated by streaking the swab over the entire sterile agar
surface. This procedure is repeated by
streaking two more times, rotating the plate approximately 60° each time to ensure an even distribution of inoculum. As a final step, the rim of the agar is
swabbed.
3. The lid may be left ajar for 3 to 5
minutes, but no more than 15 minutes, to allow for any excess surface moisture
to be absorbed before applying the drug impregnated disks.
NOTE: Extremes in inoculum density must be avoided. Never use undiluted overnight broth cultures
or other unstandardized inocula for streaking plates.
Application of Discs to Inoculated Agar Plates
1. The predetermined battery of
antimicrobial discs is dispensed onto the surface of the inoculated agar
plate. Each disc must be pressed down to
ensure complete contact with the agar surface.
Whether the discs are placed individually or with a dispensing
apparatus, they must be distributed evenly so that they are no closer than 24
mm from center to center. Ordinarily, no more than 12 discs should be placed on
one 150 mm plate or more than 5 discs on a 100 mm plate. Because some of the drug diffuses almost
instantaneously, a disc should not be relocated once it has come into contact
with the agar surface. Instead, place a
new disc in another location on the agar.
2. The plates are inverted and placed in
an incubator set to 35°C within 15 minutes
after the discs are applied. With the exception of Haemophilus spp., streptococci and
N. gonorrhoeae, the plates should not
be incubated in an increased CO2 atmosphere, because the
interpretive standards were developed by using ambient air incubation, and CO2
will significantly alter the size of the inhibitory zones of some agents.
Reading Plates and Interpreting Results
1. After 16 to 18 hours of incubation,
each plate is examined. If the plate was
satisfactorily streaked, and the inoculum was correct, the resulting zones of
inhibition will be uniformly circular and there will be a confluent lawn of
growth. If individual colonies are
apparent, the inoculum was too light and the test must be repeated. The diameters of the zones of complete
inhibition (as judged by the unaided eye) are measured, including the diameter
of the disc. Zones are measured to the nearest whole millimeter, using sliding
calipers or a ruler, which is held on the back of the inverted petri
plate. The petri plate is held a few
inches above a black, nonreflecting background and illuminated with reflected
light. If blood was added to the agar
base (as with streptococci), the zones are measured from the upper surface of
the agar illuminated with reflected light, with the cover removed. If the test organism is a Staphylococcus or Enterococcus spp., 24 hours of incubation are required for
vancomycin and oxacillin, but other agents can be read at 16 to 18 hours. Transmitted light (plate held up to light) is
used to examine the oxacillin and vancomycin zones for light growth of
methicillin- or vancomycin- resistant colonies, respectively, within apparent
zones of inhibition. Any discernable
growth within zone of inhibition is indicative of methicillin or vancomycin
resistance.
2. The zone margin should be taken as the
area showing no obvious, visible growth that can be detected with the unaided
eye. Faint growth of tiny colonies,
which can be detected only with a magnifying lens at the edge of the zone of
inhibited growth, is ignored. However,
discrete colonies growing within a clear zone of inhibition should be
subcultured, re-identified, and retested.
Strains of Proteus spp. may
swarm into areas of inhibited growth around certain antimicrobial agents. With Proteus
spp., the thin veil of swarming growth in an otherwise obvious zone of inhibition
should be ignored. When using
blood-supplemented medium for testing streptococci, the zone of growth
inhibition should be measured, not the zone of inhibition of hemolysis. With trimethoprim and the sulfonamides,
antagonists in the medium may allow some slight growth; therefore, disregard
slight growth (20% or less of the lawn of growth), and measure the more obvious
margin to determine the zone diameter.
3.
The sizes of the zones of inhibition are interpreted by referring to
Tables 2A through 2I (Zone Diameter Interpretative Standards and equivalent
Minimum Inhibitory Concentration Breakpoints) of the NCCLS M100-S12:
Performance Standards for Antimicrobial Susceptibility Testing: Twelfth
Informational Supplement, and the organisms are reported as either susceptible,
intermediate, or resistant to the agents that have been tested. Some agents may
only be reported as susceptible, since only susceptible breakpoints are given.
4.2 Dilution Methods
Dilution susceptibility testing methods are used to determine
the minimal concentration of antimicrobial to inhibit or kill the
microorganism. This can be achieved by dilution of antimicrobial in either agar
or broth media. Antimicrobials are tested in log2 serial dilutions
(two fold).
Minimum
Inhibitory Concentration (MIC)
Diffusion tests
widely used to determine the susceptibility of organisms isolated from clinical
specimens have their limitations; when equivocal results are obtained or in
prolonged serious infection e.g. bacterial endocarditis, the quantitation of
antibiotic action vis-a-vis the pathogen needs to be more precise. Also the terms ‘Susceptible’ and ‘Resistant’
can have a realistic interpretation.
Thus when in doubt, the way to a precise assessment is to determine the
MIC of the antibiotic to the organisms concerned.
There are two
methods of testing for MIC:
(a) Broth
dilution method
(b) Agar
dilution method.
Broth
Dilution Method
The Broth
Dilution method is a simple procedure for testing a small number of isolates,
even single isolate. It has the added advantage that the same tubes can
be taken for MBC tests also:
Materials
Sterile graduated pipettes of
10ml, 5ml, 2ml and 1ml Sterile capped 7.5 x 1.3 cm tubes / small screw-capped
bottles, Pasteur pipettes, overnight broth culture of test and control
organisms ( same as for disc diffusion tests), required antibiotic in powder
form (either from the manufacturer or standard laboratory accompanied by a
statement of its activity in mg/unit or
per ml. Clinical preparations should not be used for reference
technique.), required solvent for the antibiotic, sterile Distilled
Water -
500ml and suitable nutrient broth medium.
Trimethoprim and
sulphonamide testing requires thymidine free media or addition of 4% lysed
horse blood to the media
A suitable rack
to hold 22 tubes in two rows i-e 11 tubes in each row.
Stock
solution
Stock solution
can be prepared using the formula
1000
------- x V x C= W
P
Where P=Potency
given by the manufacturer in relation to the base
V= Volume in ml
required
C=Final
concentration of solution (multiples of 1000)
W= Weight of the
antimicrobial to be dissolved in the volume V
Example: For
making 10 ml solution of the strength 10,000mg/l from powder base whose potency
is 980 mg per gram,the quantities of the antimicrobials required is
W
= 1000
-------
x 10 x 10=102.04mg
980
Note:the stock
solutions are made in higher concentrations to maintain their keeping qualities
and stored in suitable aliquots at -20oC .Once taken out,they should
not be refrozen or reused.
Suggested
dilution ranges of some antimicrobials are shown in Annexure II.
Method
Prepare stock
dilutions of the antibiotic of concentrations 1000 and 100 µg/L as required
from original stock solution (10,000mg/L). Arrange two rows of 12 sterile 7.5
x1.3 cm capped tubes in the rack. In a sterile 30ml (universal) screw capped
bottle, prepare 8ml of broth containing
the concentration of antibiotic required for the first tube in each row from
the appropriate stock solution already made. Mix the contents of the universal
bottle using a pipette and transfer 2ml to the first tube in each row. Using a
fresh pipette ,add 4 ml of broth to the remaining 4 ml in the universal bottle
mix and transfer 2ml to the second tube in each row. Continue preparing
dilutions in this way but where as many as 10 or more are required the series
should be started again half the way down. Place 2ml of antibiotic free broth
to the last tube in each row. Inoculate one row with one drop of an overnight
broth culture of the test organism diluted approximately to 1 in 1000 in a
suitable broth and the second row with the control organism of known
sensitivity similarly diluted. The result of the test is significantly affected by the size of the
inoculum.The test mixture should contain 106 organism/ml.If the
broth culture used has grown poorly,it may be necessary to use this undiluted.
Incubate tubes for 18 hours at 37oC. Inoculate a tube containing 2ml
broth with the organism and keep at +4oC in a refrigerator overnight
to be used as standard for the determination of complete inhibition.
Calculations
for the preparation of the original dilution.
This often presents problems to those unaccustomed
to performing these tests. The following method advocated by Pamela M
Waterworth is presented. Calculate the total volume required for the first
dilution. Two sets of dilution are being prepared (one for the test and one for
the control), each in 2ml volumes i-e a total of 4 ml for each concentration as
4ml is required to make the second dilution, the total requirement is 8ml. Now
calculate the total amount of the antibiotic required for 8ml. For 64 g/l
concentration, 8x64mg/l =512µg in 8 ml. Place a decimal point after the first
figure (5.12) and take this volume in ml (i.e 5.12 ml) of the dilution below
512mg/l and make upto 8ml with broth. In this example given above, the series
has to be started again mid way at 2 mg/l which would be obtained in the same
way:
8ml of 2mg/l=16µg in 8ml.
1.6 ml of 10 mg/ l + 6.4 ml of
broth.
Reading
of result
MIC is expressed
as the lowest dilution, which inhibited growth judged by lack of turbidity in
the tube.
Because very
faint turbidity may be given by the inoculum itself, the inoculated tube kept
in the refrigerator overnight may be used as the standard for the determination
of complete inhibition.
Standard strain
of known MIC value run with the test is used as the control to check the
reagents and conditions.
Minimum
Bactericidal Concentrations(MBC)
The main
advantage of the ‘Broth dilution’ method for the MIC determination lies in the
fact that it can readily be converted to determine the MBC as well.
Method
Dilutions and
inoculations are prepared in the same manner as described for the determination
of MIC. The control tube containing no antibiotic is immediately subcultured
(Before incubation) by spreading a loopful evenly over a quarter of the plate
on a medium suitable for the growth of the test organism and incubated at 37oC
overnight. The tubes are also incubated overnight at 37oC. Read the
MIC of the control organism to check that the drug concentrations are correct.
Note the lowest concentration inhibiting growth of the organisms and record
this as the MIC. Subculture all tubes not showing visible growth in the same
manner as the control tube described above and incubate at 37oC
overnight. Compare the amount of growth from the control tube before
incubation,which represents the original inoculum. The test must include a
second set of the same dilutions inoculated with an organism of known
sensitivity .These tubes are not subcultured; the purpose of the control is to
confirm by its MIC that the drug level is correct,whether or not this organism
is killed is immaterial.
Reading of result
These
subcultures may show
- Similar number of colonies- indicating bacteriostasis
only.
·
A reduced
number of colonies-indicating a partial
or slow bactericidal activity.
·
No growth- if
the whole inoculum has been killed
·
The highest
dilution showing at least 99% inhibition is taken as MBC
Micro-broth
dilution test
This test uses
double-strength Müeller-Hinton broth, 4X strength antibiotic solutions prepared
as serial two-fold dilutions and the test organism at a concentration of 2x106/ml.
In a 96 well plate, 100 ml of double-strength MHB, 50 ml each of the antibiotic dilutions and the
organism suspension are mixed and incubated at 35°C for 18-24 hours. The lowest concentration
showing inhibition of growth will be considered the MIC of the organism.
Reading
of result
MIC is expressed
as the highest dilution which inhibited growth judged by lack of turbidity in
the tube. Because very faint turbidity may be given by the inoculum itself,the
inoculated tube kept in the refrigerator overnight may be used as the standard
for the determination of complete inhibition. Standard strain of known MIC, run
with the test is used as the control to check the reagents and conditions.
The
Agar dilution Method
Agar dilutions
are most often prepared in petri dishes and have advantage that it is
possible to test several organisms on each plate .If only one
organism is to be tested e.g M.tuberculosis,the
dilutions can be prepared in agar slopes but it will then be necessary to
prepare a second identical set to be inoculated with the control organism.The
dilutions are made in a small volume of water and added to agar which has been
melted and cooled to not more than 60oC.Blood may be added and if
‘chocolate agar’ is required,the medium must be heated before the antibiotic is
added.
It would be
convenient to use 90 mm diameter petri dishes and add
one ml of
desired drug dilutions to 19 ml of broth.The factor of agar dilution must be
allowed for in the first calculation as follows.
final volume of
medium in plate = 20 ml
Top antibiotic
concentrations =
64mg/l
Total amount of
drug =
1280µg to be added to1 ml of water
2ml of 1280 µg
/ml will be required to start the dilution =
2560µg in 2 ml
=
1.28ml of 2000µg /ml ±
0.72 ml of water.
1
ml of this will be added to 19 ml agar.
(Note stock
dilution of 2000µg /ml is required for this range of MIC)
The quickest
way to prepare a range of dilutions in agar is as follows:
Label a sterile
petri dish on the base for each concentration required. Prepare the dilutions
in water placing 1 ml of each in the appropriate dish. One ml water is added to
a control plate. Pipette 19 ml melted agar, cooled to 55oC to each
plate and mix thoroughly. Adequate mixing is essential and if sufficient
technical expertise is not available for the skilled manipulation, it is
strongly recommended that the agar is first measured into stoppered tubes or
universal containers and the drug dilution added to these and mixed by inversion
before pouring into petri dishes. After the plates have set they should be well
dried at 37oC with their lids tipped for 20 to 30 minutes in an
incubator. They are then inoculated either with a multiple inoculator as spots
or with a wire loop or a platinum loop calibrated to deliver 0.001ml spread
over a small area. In either case the culture should be diluted to contain 105
to 106 organisms per ml. With ordinary fast growing organisms, this
can be obtained approximately by adding 5 µl of an overnight broth culture to
5ml broth or peptone water.
It is possible
to test spreading organism such as P.mirabilis
by this method either by cutting ditches in the agar between the inocula, or by
confining each with small glass or porcelain cylinders pressed into the agar.
Although swarming of P.mirabilis can be prevented by the use of higher
concentration of agar in the medium, this is not recommended for determination
of MIC because of the difficulty of ensuring adequate mixing of the drug with
this very viscous medium. Selective media should not be used and electrolyte
deficient media will give false results because of the effect of variation in
the salt content on the action of many antibiotics.
Reading
of results
The antibiotic concentration of the first
plate showing ³ 99% inhibition is taken as the MIC for the organism.
4.3
Dilution and Diffusion
E
test also known as the epsilometer test is an ‘exponential gradient’ testing
methodology where ‘E’ in E test refers to the
Greek symbol epsilon (e).The E test(AB Biodisk) which is a quantitative method for
antimicrobial susceptibility testing applies
both the dilution of antibiotic and diffusion of antibiotic into the medium.. A
predefined stable antimicrobial gradient is present on a thin inert carrier
strip. When this E test strip is applied onto an inoculated agar plate, there
is an immediate release of the drug. Following incubation , a symmetrical
inhibition ellipse is produced. The intersection of the inhibitory zone edge
and the calibrated carrier strip indicates the MIC value over a wide concentration range (>10 dilutions) with
inherent precision and accuracy .
E test
can be used
to determine MIC
for fastidious organisms
like S. pneumoniae,
ß-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also be used for Nonfermenting Gram Negative bacilli (NFGNB) for eg-Pseudomonas sp. and Burkholderia pseudomallei.
Resistance of
major consequence may be detected for e.g., the test is very useful in
detecting glycopeptide resistant Enterococci (GRE) and glycopeptide
intermediate S.aureus (GISA) and slow growing pathogens such as Mycobacterium
tuberculosis. Further it can be used for detection of extended spectrum
beta lactamases (ESBL). In conclusion E test is a simple, accurate and reliable
method to determine the MIC for a wide spectrum of infectious agents.
